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Chapter 19 Chapter 19 Presentation Transcript

  • Chapter 19 Spectrophotometry :Instruments & Application
  • 19.1 The Spectrophotometer –1Remote sensing of airborne bacteria: Optical fiber coated with antibodies to detect spores of a specific bacterium
  • 19.1 The Spectrophotometer -11) Spectrophotometerb) Single-beame) Double-beam
  • 19.1 The Spectrophotometer -2
  • 19.1 The Spectrophotometer -3
  • 19.1 The Spectrophotometer –4• Light source – Tungsten lamp: Vis. near IR (320 nm~2500 nm) – Deuterium are lamp: UV (200~400 nm) – Electric discharge lamp + Hg(g) or Xenon: Vis & UV – Globar (silicon carbide rod): IR (5000~200 cm-1) – Laser: intense monochromatic sources.
  • 19.1 The Spectrophotometer -5
  • 19.1 The Spectrophotometer -6• MonochromatorConsists:• lenses or mirrors: focus the radiation• entrance and exit slits: restrict unwanted and control the spectral purity of radiation.• dispersing medium: separate the λ of polychromatic radiation from the source. (a) prism and (b) diffraction grating see Fig 19-2
  • 19.1 The Spectrophotometer -71) Monochromator a. entrance slit b. collimating mirror or lens c. a prism or grating d. focal plane e. exit slit
  • 19.1 The Spectrophotometer -8• Monochromator Interference of adjacent waves that are (a) 0°, (b) 90 °, and © 180 ° out of phase
  • 19.1 The Spectrophotometer -9• Monochromator nλ = a – b (n = ±1 first-order….) Grating equation : nλ = d (sinθ – sinφ ) Filters: select a desired wavelength
  • 19.1 The Spectrophotometer -10• MonochromatorChoosing the bandwidth: exit slit widthResolution trade-off Signal
  • 19.1 The Spectrophotometer -101) Detector Convert radiant energy (photons) into an electrical signal Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.
  • 19.1 The Spectrophotometer -11 3) Detector Detector response depends on the λ of the incident photons.
  • 19.1 The Spectrophotometer -12Photomultiplier tube: very sensitive detector
  • 19.1 The Spectrophotometer -13Photodiode array spectrophotometer :records the entire spectrum at once. vs. Dispersive spectrophotometer: one λ at a time • speed (~1s/spetrum) • excellent λ repeatability • measure λs simultaneously • relatively insensitive to errors from stray light • relatively poor resolution (1~3 nm) vs 0.1nm
  • 19.1 The Spectrophotometer -14 diode array spectrophotometer
  • 19.2 Analysis of a mixture -1• Absorbance of a mixture : A = exb[X] + eyb[Y] + …
  • 19.2 Analysis of a mixture -2• Isosbestic points : for rxn: X → Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn. Ex: HIn  In- + H+
  • 19.2 Analysis of a mixture -3Why isosbestic point? A 465 = ε 465 [ HIn ] HIn [ ] A 465 = ε 465 In − In − when [ HIn ] = [ In ] ⇒ ε − 465 HIn = ε 465 = ε 465 In − ∴ For a mixture : HIn In − [ ] A 465 = ε 465 b [ HIn ] + ε 465 b In − = ε 465 b ( [ In ] + [ HIn ] ) − −
  • 19.3 Spectrophotometric Titrations -1 apotransferrin + 2Fe3+  (Fe3+)2transferrin colorless red (465 nm)
  • 19.3 Spectrophotometric Titrations-2 Ferric nitrilotriacetate [used to avoid Fe(OH)3 ]
  • 19.3 Spectrophotometric Titrations-3 ex.at p.408 Correcting A for the effect of dilution 125 μL ferric nitrilotriancetate 2 mL apotransferrin A = 0.260 A corrected = ?
  • 19.4 What happens when a molecule absorbs light ? 1) Absorbing species : M + hν → M* (lifetime : 10-8 ~ 10-9 sec) Relaxation processes : • M* → M + heat (most common) • M* → new species (photochemical reaction) • M* → M + hν (fluorescence, phosphorescence)
  • 19.4• Geometry of formaldehyde
  • 19.4 What happens when a molecule absorbs light ? • Types of absorbing electrons Consider formaldehyde: three types of molecular orbitals • =σ H C O ×=π H =n
  • 19.4 What happens absorbs light ? MO of CH3CO
  • 19.4 What happens when a molecule absorbs light ? Four types of electronic transitions σ* π* E n 200~700 nm π 150~250 nm σ < 125 nm
  • 19.4 What happens when a molecule absorbs light ? -5 1) Singlet / Triplet excited states ground excited excited singlet state singlet (S1) triplet (T1) E: T1 < S1
  • 19.4 What happens when a molecule absorbs light ? -61) Electronic transition of formaldehyden → π* (T1), absorption of light at λ = 397 nm green-yellown → π* (S1), absorption of light at λ = 355 nm colorless (more probable)
  • 19.4 What happens when absorbs light ?• Vibrational & Rotational states of CH3CO (IR and microwave radiation)
  • 19.4 a molecule absorbs light 1) What happens to absorbed energy
  • 19.4 a molecule absorbs light 7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis.  Photoluminescence : • Fluorescence : S1 → S0, no change in electron spin. (< 10-5 s) • Phosphorescence : T1 → S0, with a change in electron spin. (10-4~102 s) b. Chemiluminescence : Chemical reaction (not initiated by light) release energy in the form of light. ex : firefly.
  • 19.4 a molecule absorbs light 1) In which your class really shines ? emission spectrum
  • 19.4 a molecule absorbs light1) Absorption & Emission Spectra
  • 19.5 Luminescence in analytical chemistry1) Instrument • .hνout (photon) • heat hνin • breaking a chemical bond
  • 19.5 Luminescence• I = kPoC  incident radiation sensitivity  by P0  or C 6) more sensitive than Absorption
  • 19.5 Luminescence4) Fluorimetric Assay of Selenium in Brazil Nuts – Se is a trace element essential to life: destruct ROOH (free radical) – Derivatized: – Self-absorption: quench
  • 19.5 Luminescence5) Immunoassarys  employ anitbody to detect analyte. Ex: ELISA
  • 19.5 Luminescence• pregnancy test. sensitive to < 1 ng of analyte• Enviromental Analysis. (ppm) or (ppt) pesticides, industrial chemicals, & microbialtoxins.
  • 19.5 Luminescence• Environmental Analysis
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